Inbred maize line NP2315

ABSTRACT

The invention relates to an inbred maize line, designated NP2315, to the seeds of inbred maize line NP2315, to the plants of inbred maize line NP2315, and to methods for producing a maize plant by crossing plants of maize inbred line NP2315 with itself or with another maize plant. The invention also relates to maize plants of inbred line NP2315 containing a transgene produced by transforming said plants with said transgene.

FIELD OF THE INVENTION

This invention is in the field of maize breeding, specifically relatingto an inbred maize line designated NP2315.

BACKGROUND OF THE INVENTION

The goal of plant breeding is to combine in a single variety or hybridvarious desirable traits. For field crops, these traits may includeresistance to diseases and insects, tolerance to heat and drought,reducing the time to crop maturity, greater yield, and better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination and stand establishment, growthrate, maturity, and plant and ear height, is important.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant. Plants that have been self-pollinated and selectedfor type for many generations become homozygous at almost all gene lociand produce a uniform population of true breeding progeny. A crossbetween two different homozygous lines produces a uniform population ofhybrid plants that may be heterozygous for many gene loci. A cross oftwo plants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Maize (Zea mays L.), often referred to as corn in the United States, canbe bred by both self-pollination and cross-pollination techniques. Maizehas separate male and female flowers on the same plant, located on thetassel and the ear, respectively. Natural pollination occurs in maizewhen wind blows pollen from the tassels to the silks that protrude fromthe tops of the ears.

A reliable method of controlling male fertility in plants offers theopportunity for improved plant breeding. This is especially true fordevelopment of maize hybrids, which relies upon some sort of malesterility system. There are several options for controlling malefertility available to breeders, such as: manual or mechanicalemasculation (or detasseling), cytoplasmic male sterility, genetic malesterility, gametocides and the like.

Hybrid maize seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male) and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent inmaize plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile maize and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 and chromosomal translocations as described inU.S. Pat. Nos. 3,861,709 and 3,710,511, the disclosures of which arespecifically incorporated herein by reference. There are many othermethods of conferring genetic male sterility in the art, each with itsown benefits and drawbacks. These methods use a variety of approachessuch as delivering into the plant a gene encoding a cytotoxic substanceassociated with a male tissue specific promoter or an antisense systemin which a gene critical to fertility is identified and an antisense tothat gene is inserted in the plant (EPO 89/3010153.8 and WO 90/08828).

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, Glenn R., U.S. Pat. No. 4,936,904, which isincorporated herein by reference). Application of the gametocide, timingof the application and genotype specificity often limit the usefulnessof the approach.

The use of male sterile inbreds is but one factor in the production ofmaize hybrids. The development of maize hybrids requires, in general,the development of homozygous inbred lines, the crossing of these lines,and the evaluation of the crosses. Pedigree breeding and recurrentselection breeding methods are used to develop inbred lines frombreeding populations. Breeding programs combine the genetic backgroundsfrom two or more inbred lines or various other germplasm sources intobreeding pools from which new inbred lines are developed by selfing andselection of desired phenotypes. The new inbreds are crossed with otherinbred lines and the hybrids from these crosses are evaluated todetermine which of those have commercial potential. Plant breeding andhybrid development are expensive and time-consuming processes.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding five or more generations ofselfing and selection is practiced: F1 to F2; F3 to F4; F4 to F5, etc.

Recurrent selection breeding can be used to improve populations ofeither self or cross-pollinating crops. Recurrent selection can be usedto transfer a specific desirable trait from one inbred or source to aninbred that lacks the trait. This can be accomplished, for example, byfirst a superior inbred (recurrent parent) to a donor inbred(non-recurrent parent), that carries the appropriate gene(s) for thetrait in question. The progeny of this cross is then mated back to thesuperior recurrent parent followed by selection in the resultant progenyfor the desired trait to be transferred from the non-recurrent parent.After five or more backcross generations with selection for the desiredtrait, the progeny will be homozygous for loci controlling thecharacteristic being transferred, but will be like the superior parentfor essentially all other genes. The last backcross generation is, thenselfed to give pure breeding progeny for the gene(s) being transferred.A hybrid developed from inbreds containing the transferred gene(s) isessentially the same as a hybrid developed form the same inbreds withoutthe transferred genes. As the varieties developed using recurrentselection breeding contain almost all of the characteristics of therecurrent parent, selecting a superior recurrent parent is desirable.

A single cross maize hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F1. In thedevelopment of commercial hybrids only the F1 hybrid plants are sought.Preferred F1 hybrids are more vigorous than their inbred parents. Thishybrid vigor, or heterosis, can be manifested in many polygenic traits,including increased vegetative growth and increased yield.

The development of a maize hybrid involves three steps: (1) theselection of plants from various germplasm pools for initial breedingcrosses; (2) the selfing of the selected plants from the breedingcrosses for several generations to produce a series of inbred lines,which, although different from each other, breed true and are highlyuniform; and (3) crossing the selected inbred lines with differentinbred lines to produce the hybrid progeny (F1). During the inbreedingprocess in maize, the vigor of the lines decreases. Vigor is restoredwhen two different inbred lines are crossed to produce the hybridprogeny (F1). An important consequence of the homozygosity andhomogeneity of the inbred lines is that the hybrid between a definedpair of inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

A single cross hybrid is produced when two inbred lines are crossed toproduce the F1 progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F1 hybridsare crossed again (A×B)×(C×D). Much of the hybrid vigor exhibited by F1hybrids is lost in the next generation (F2). Consequently, seed fromhybrids is not used for planting stock.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female parent. Incomplete removal or inactivation of thepollen provides the potential for self-pollination. This inadvertentlyself-pollinated seed may be unintentionally harvested and packaged withhybrid seed. Once the seed is planted, it is possible to identify andselect these self-pollinated plants. These self-pollinated plants willbe genetically equivalent to the female inbred line used to produce thehybrid. Typically these self-pollinated plants can be identified andselected due to their decreased vigor. Female selfs are identified bytheir less vigorous appearance for vegetative and/or reproductivecharacteristics, including shorter plant height, small ear size, ear andkernel shape, cob color, or other characteristics.

Identification of these self-pollinated lines can also be accomplishedthrough molecular marker analyses. See, “The Identification of FemaleSelfs in Hybrid Maize: A Comparison Using Electrophoresis andMorphology”, Smith, J. S. C. and Wych, R. D., Seed Science andTechnology 14, pp. 1–8 (1995), the disclosure of which is expresslyincorporated herein by reference. Through these technologies, thehomozygosity of the self-pollinated line can be verified by analyzingallelic composition at various loci along the genome. Those methodsallow for rapid identification of the invention disclosed herein. Seealso, “Identification of A typical Plants in Hybrid Maize Seed byPostcontrol and Electrophoresis” Sarca, V. et al., Probleme de GeneticaTeoritca si Aplicata Vol. 20 (1) p. 29–42.

As is readily apparent to one skilled in the art, the foregoingdescribes only two of the various ways by which the inbred can beobtained by those looking to use the germplasm. Other means areavailable, and the above examples are illustrative only.

Maize is an important and valuable field crop. Thus, a continuing goalof plant breeders is to develop high-yielding maize hybrids that areagronomically sound based on stable inbred lines. The reasons for thisgoal are obvious: to maximize the amount of grain produced with theinputs used and minimize susceptibility of the crop to pests andenvironmental stresses. To accomplish this goal, the maize breeder mustselect and develop superior inbred parental lines for producing hybrids.This requires identification and selection of genetically uniqueindividuals that occur in a segregating population. The segregatingpopulation is the result of a combination of crossover events plus theindependent assortment of specific combinations of alleles at many geneloci that results in specific genotypes. The probability of selectingany one individual with a specific genotype from a breeding cross isinfinitesimal due to the large number of segregating genes and theunlimited recombinations of these genes, some of which may be closelylinked. However, the genetic variation among individual progeny of abreeding cross allows for the identification of rare and valuable newgenotypes. These new genotypes are neither predictable nor incrementalin value, but rather the result of manifested genetic variation combinedwith selection methods, environments and the actions of the breeder.Thus, even if the entire genotypes of the parents of the breeding crosswere characterized and a desired genotype known, only a few, if any,individuals having the desired genotype may be found in a largesegregating F2 population. Typically, however, neither the genotypes ofthe breeding cross parents nor the desired genotype to be selected isknown in any detail. In addition, it is not known how the desiredgenotype would react with the environment. This genotype by environmentinteraction is an important, yet unpredictable, factor in plantbreeding. A breeder of ordinary skill in the art cannot predict thegenotype, how that genotype will interact with various climaticconditions or the resulting phenotypes of the developing lines, exceptperhaps in a very broad and general fashion. A breeder of ordinary skillin the art would also be unable to recreate the same line twice from thevery same original parents, as the breeder is unable to direct how thegenomes combine or how they will interact with the environmentalconditions. This unpredictability results in the expenditure of largeamounts of research resources in the development of a superior new maizeinbred line.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred maize line,designated NP2315, having excellent Eye Spot and Northern Leaf Blightresistance relative to A619, a relative maturity of approximately 95–105days based on the Comparative Relative Maturity Rating System forharvest moisture of grain, good Northern Corn Leaf Blight andHelminthosporium Leaf Blight resistance relative to A619, pale purplesilk ends 3 days after emergence, and adapted to the Northern Cornbeltregions of the United States. This invention relates to the seeds ofinbred maize line NP2315, to the plants of inbred maize line NP2315, todescendants of NP2315, and to methods for producing a maize plant bycrossing the inbred line NP2315 with itself or another maize line. Thisinvention further relates to hybrid maize seeds and plants produced bycrossing the inbred line NP2315 with another maize line.

The invention is also directed to inbred maize line NP2315 into whichone or more specific, single gene traits, for example transgenes, havebeen transformed and/or introgressed.

The invention is also directed to a NP2315-derived maize plant, or partsthereof, wherein at least one ancestor of the NP2315-derived maize plantis the inbred maize line NP2315, wherein the NP2315-derived maize plantexpresses a combination of at least two NP2315 traits selected from thegroup consisting of: a relative maturity of approximately 95–105 daysbased on the Comparative Relative Maturity Rating System for harvestmoisture of grain, good Northern Corn Blight resistance andHelminthosporium Leaf Blight resistance relative to A619, pale purplessilk ends 3 days after emergence, and adapted to the Northern Cornbeltregions of the United States.

DETAILED DESCRIPTION OF THE INVENTION

Inbred maize lines are typically developed for use in the production ofhybrid maize lines. Inbred maize lines need to be highly homogeneous,homozygous and reproducible to be useful as parents of commercialhybrids. There are many analytical methods available to determine thehomozygotic and phenotypic stability of these inbred lines.

The oldest and most traditional method of analysis is the observation ofphenotypic traits. The data is usually collected in field experimentsover the life of the maize plants to be examined. Phenotypiccharacteristics most often observed are for traits associated with plantmorphology, ear and kernel morphology, insect and disease resistance,maturity, and yield.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), and Simple SequenceRepeats (SSRs) which are also referred to as Microsatellites.

Some of the most widely used of these laboratory techniques are IsozymeElectrophoresis and RFLPs as discussed in Lee, M., “Inbred Lines ofMaize and Their Molecular Markers,” The Maize Handbook,(Springer-Verlag, New York, Inc. 1994, at 423–432). IsozymeElectrophoresis is a useful tool in determining genetic composition,although it has relatively low number of available markers and the lownumber of allelic variants among maize inbreds. RFLPs have the advantageof revealing an exceptionally high degree of allelic variation in maizeand the number of available markers is almost limitless. Maize RFLPlinkage maps have been rapidly constructed and widely implemented ingenetic studies. One such study is described in Boppenmaier, et al.,“Comparisons among strains of inbreds for RFLPs”, Maize GeneticsCooperative Newsletter, 65:1991, pg. 90. This study used 101 RFLPmarkers to analyze the patterns of 2 to 3 different deposits each offive different inbred lines. The inbred lines had been selfed from 9 to12 times before being adopted into 2 to 3 different breeding programs.It was results from these 2 to 3 different breeding programs thatsupplied the different deposits for analysis. These five lines weremaintained in the separate breeding programs by selfing or sibbing androgueing off-type plants for an additional one to eight generations.After the RFLP analysis was completed, it was determined the five linesshowed 0–2% residual heterozygosity. Although this was a relativelysmall study, it can be seen using RFLPs that the lines had been highlyhomozygous prior to the separate strain maintenance.

The production of hybrid maize lines typically comprises planting inpollinating proximity seeds of, for example, inbred maize line NP2315and of a different inbred parent maize plant, cultivating the seeds ofinbred maize line NP2315 and of said different inbred parent maize plantinto plants that bear flowers, emasculating the male flowers of inbredmaize line NP2315 or the male flowers of said different inbred parentmaize plant to produce an emasculated maize plant, allowingcross-pollination to occur between inbred maize line NP2315 and saiddifferent inbred parent maize plant and harvesting seeds produced onsaid emasculated maize plant. The harvested seed are grown to producehybrid maize plants.

Inbred maize line NP2315 can be crossed to inbred maize lines of variousheterotic group (see e.g. Hallauer et al. (1988) in Corn and CornImprovement, Sprague et al, eds, chapter 8, pages 463–564) for theproduction of hybrid maize lines.

TABLE I VARIETY DESCRIPTION INFORMATION Inbred maize line NP2315 iscompared to inbred A619 INBRED NP2315 INBRED A619 MATURITY Days HeatDays Heat Units Units From emergence to 50% of plants in silk 070 1385.5068 1319.9 From emergence to 50% of plants in pollen 069 1348.1 0661276.3 From 10% to 90% pollen shed 003 0152.6 003 0115.3 PLANT Std DevSample Std Dev Sample Size Size cm Plant Height (to tassel tip) 159.57.0 8 182.3 19.9 8 cm Ear Height (to base of top ear node) 053.1 6.6 8044.3 6.2 8 cm Length of Top Ear Internodenode 13.0 1.2 7 14.6 0.9 7Average Number of Tillers 0 0 7 0.1 0.3 7 Average Number of Ears perStalk 1.4 0.6 6 1.2 0.3 6 Anthocyanin of Brace Roots: 3 2 1 = Absent 2 =Faint 3 = Moderate 4 = Dark LEAF Std Dev Sample Std Dev Sample Size SizeCm Width of Ear Node Leaf 010.4 0.9 8 008.9 0.6 8 cm Length of Ear NodeLeaf 061.6 5.1 8 068.2 8.2 8 Number of leaves above top ear 5 0.7 8 60.4 8 degrees Leaf Angle 44 17.0 8 46 22.2 7 (measure from 2nd leafabove ear at anthesis to stalk above leaf) Leaf Color 02 (Munsell code5GY 03 (Munsell code 5GY 5/4) 4/4) Leaf Sheath Pubescence 6 2 (Rate onscale from 1 = none to 9 = like peach fuzz) Marginal Waves 5 6 (Rate onscale from 1 = none to 9 = many) Longitudinal Creases 6 5 (Rate on scalefrom 1 = none to 9 = many) TASSEL Number of Primary Lateral Branches 73.9 8 6 2.7 8 Branch Angle from Central Spike 37 12.8 7 56 9.5 5 CmTassel Length 40.7 3.7 7 41.4 5.0 7 (from top leaf collar to tassel tip)Pollen Shed 5 6 (Rate on scale from 0 = male sterile to 9 = heavy shed)Anther Color 05 (Munsell code 5GY 05 (Munsell code 2.5GY 8/6) 8/6) GlumeColor 02 (Munsell code 5GY 03 (Munsell code 7.5GY 5/6) 5/6) Bar Glumes(Glume Bands): 1 = Absent 2 2 2 = Present EAR (Unhusked Data) Silk Color(3 days after emergence) 26 (Munsell code) 05 (Munsell code 2.5GY 8/6)Fresh Husk Color(25 days after 50% 05 (Munsell code 5GY 05 (Munsell code5GY silking) 7/6) 8/6) Dry Husk Color (65 days after 50% 22 (Munsellcode 2.5Y 22 (Munsell code 2.5Y silking) 8/4) 8/4) Position of Ear atDry Husk Stage: 3 1 1 = Upright 2 = Horizontal 3 = Pendent HuskTightness 7 6 (Rate on scale from 1 = very loose to 9 = very tight) HuskExtension (at harvest): 2 2 1 = Short (ears exposed) 2 = Medium (<8 cm)3 = Long (8–10 cm beyond ear tip) 4 = Very long (>10 cm) EAR (Husked EarData) Std Dev Sample Std Dev Sample Size Size Cm Ear Length 13.6 0.7 813.7 1.6 8 mm Ear Diameter at mid-point 37.8 1.1 8 44.9 2.0 7 gm EarWeight 95.9 27.3 8 115.0 20.4 7 Number of Kernel Rows 16 0.8 8 15 0.2 7Kernel Rows: 1 = Indistinct 2 = Distinct 2 2 Row Alignment: 1 1 1 =Straight 2 = Slightly Curved 3 = Spiral cm Shank Length 10.9 1.7 7 11.43.4 7 Ear Taper: 1 = Slight 2 = Average 2 2 3 = Extreme KERNEL (Dried)Std Dev Sample Std Dev Sample Size Size mm Kernel Length 09.7 0.6 7 10.80.9 7 mm Kernel Width 7.4 0.5 7 08.6 0.5 7 mm Kernel Thickness 05.2 0.57 04.4 1.1 7 % Round Kernels (Shape Grade) 57.0 4.9 6 38.8 24.7 6Aleurone Color Pattern: 1 = Homozygous 2 = Segregating 1 1 AleuroneColor 19 (Munsell code) 19 (Munsell code) Hard Endosperm Color 06(Munsell code 2.5Y 06 (Munsell code 2.5Y 8/10) 8/8) Endosperm Type: 3 31 = Sweet (su1) 2 = Extra Sweet (sh2) 3 = Normal Starch Gm Weight per100 Kernels (unsized 27.7 1.5 7 30.4 2.1 7 sample) COB Std Dev SampleStd Dev Sample Size Size mm Cob Diameter at mid-point 23.5 1.6 8 26.50.8 8 Cob Color 19 (Munsell code ) 19 (Munsell code) DISEASE RESISTANCE(1 = most susceptible to 9 = most resistant) Eye Spot (Kabatiella zeae)8 5 Northern Leaf Blight 8 Mixed 5 Mixed Inoc. Inoc. Gray Leaf SpotCommon Rust Helminthosporium Leaf Blight 8 5 INSECT RESISTANCE(Rate from1 = most susceptible to 9 = most resistant) European CornBorer(Osstrinia nubilalis) 1^(st) Generation (Typically Whorl LeafFeeding) 2^(nd) Generation Corn Borer AGRONOMIC TRAITS Stay Green (at 65days after anthesis) 7 7 (rate on scale from 1 = worst to 9 = excellent)% Dropped Ears (at 65 days after anthesis) 0 0 % Pre-anthesis Brittlesnapping 0 0 % Pre-anthesis Root Lodging 0 3 % Post-anthesis RootLodging 0 0 (at 65 days after anthesis) Kg/ha Yield of Inbred Per Se (at12–13% grain moisture) In interpreting the foregoing color designations,reference may be made to the Munsell Glossy Book of Colr, a standardcolor reference. Color codes: 1. light green, 2. medium green, 3. darkgreen, 4. very dark green, 5. green-yellow, 6. pale yellow, 7. yellow,8. yellow-orange, 9. salmon, 10. pink-orange, 11. pink, 12. light red,13 cherry red. 14. red, 15. red and white, 16. pale purple, 17. purple,18. colorless, 19. white, 20, white capped, 21. buff, 22. tan, 23.brown, 24. bronze, 25. variegated, 26. other.Comments:

-   1) Heat Units per day were calculated using the standard formula:    HU={MaxTemp (86)+Min Temp (50)]/2−50.-   2) Data for this exhibit was collected at London, Ontario, Canada,    MN and WI.-   3) Large standard deviations are probably due to environmental    factors at each individual location where the variety was observed.    Since the varieties reported in this exhibit are inbreds, the    response to the environment is probably more pronounced than a    hybrid or a combination of these inbred lines. Any stress at    specific times during the growing season could influence results.-   4) The anther of NP2315 appears to have a slight pale purple shade,    whereas this coloring is absent in A619.-   5) Glume bars of NP2315 and A619 are green/yellow, whereas some    glume bars of A619 have a pale purple shade.-   6) The silk color of NP2315 is green/yellow (2.5GY 8/8) with pale    purple shaded ends (5RP 5/6).-   7) The Disease and Insect Ratings were taken in MN in 2001 and 2002.

The invention also encompasses plants of inbred maize line NP2315 andparts thereof further comprising one or more specific, single genetraits which have been introgressed into inbred maize line NP2315 fromanother maize line. Preferably, one or more new traits are transferredto inbred maize line NP2315, or, alternatively, one or more traits ofinbred maize line NP2315 are altered or substituted. The transfer (orintrogression) of the trait(s) into inbred maize line NP2315 is forexample achieved by recurrent selection breeding, for example bybackcrossing. In this case, inbred maize line NP2315 (the recurrentparent) is first crossed to a donor inbred (the non-recurrent parent)that carries the appropriate gene(s) for the trait(s) in question. Theprogeny of this cross is then mated back to the recurrent parentfollowed by selection in the resultant progeny for the desired trait(s)to be transferred from the non-recurrent parent. After three, preferablyfour, more preferably five or more generations of backcrosses with therecurrent parent with selection for the desired trait(s), the progenywill be heterozygous for loci controlling the trait(s) beingtransferred, but will be like the recurrent parent for most or almostall other genes (see, for example, Poehlman & Sleper (1995) BreedingField Crops, 4th Ed., 172–175; Fehr (1987) Principles of CultivarDevelopment, Vol. 1: Theory and Technique, 360–376).

The laboratory-based techniques described above, in particular RFLP andSSR, are routinely used in such backcrosses to identify the progenieshaving the highest degree of genetic identity with the recurrent parent.This permits to accelerate the production of inbred maize lines havingat least 90%, preferably at least 95%, more preferably at least 99%genetic identity with the recurrent parent, yet more preferablygenetically identical to the recurrent parent, and further comprisingthe trait(s) introgressed from the donor patent. Such determination ofgenetic identity is based on molecular markers used in thelaboratory-based techniques described above. Such molecular markers arefor example those known in the art and described in Boppenmaier, et al.,“Comparisons among strains of inbreds for RFLPs”, Maize GeneticsCooperative Newsletter (1991) 65, pg. 90, or those available from theUniversity of Missouri database and the Brookhaven laboratory database.The last backcross generation is then selfed to give pure breedingprogeny for the gene(s) being transferred. The resulting plants haveessentially all of the morphological and physiological characteristicsof inbred maize line NP2315, in addition to the single gene trait(s)transferred to the inbred. The exact backcrossing protocol will dependon the trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the trait beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired trait has been successfullytransferred.

Many traits have been identified that are not regularly selected for inthe development of a new inbred but that can be improved by backcrossingtechniques or genetic transformation. Examples of traits transferred toinbred maize line NP2315 include, but are not limited to, waxy starch,herbicide tolerance, resistance for bacterial, fungal, or viral disease,insect resistance, enhanced nutritional quality, improved performance inan industrial process, altered reproductive capability, such as malesterility or male fertility, yield stability and yield enhancement.Other traits transferred to inbred maize line NP2315 are for theproduction of commercially valuable enzymes or metabolites in plants ofinbred maize line NP2315.

Traits transferred to maize inbred line NP2315 are naturally occurringmaize traits, which are preferably introgressed into inbred maize lineNP2315 by breeding methods such as backcrossing, or are heterologoustransgenes, which are preferably first introduced into a maize line bygenetic transformation using genetic engineering and transformationtechniques well known in the art, and then introgressed into inbred lineNP2315. Alternatively a heterologous trait is directly introduced intoinbred maize line NP2315 by genetic transformation. Heterologous, asused herein, means of different natural origin or represents anon-natural state. For example, if a host cell is transformed with anucleotide sequence derived from another organism, particularly fromanother species, that nucleotide sequence is heterologous with respectto that host cell and also with respect to descendants of the host cellwhich carry that gene. Similarly, heterologous refers to a nucleotidesequence derived from and inserted into the same natural, original celltype, but which is present in a non-natural state, e.g. a different copynumber, or under the control of different regulatory sequences. Atransforming nucleotide sequence may comprise a heterologous codingsequence, or heterologous regulatory sequences. Alternatively, thetransforming nucleotide sequence may be completely heterologous or maycomprise any possible combination of heterologous and endogenous nucleicacid sequences.

A transgene introgressed into maize inbred line NP2315 typicallycomprises a nucleotide sequence whose expression is responsible orcontributes to the trait under the control of a promoter appropriate forthe expression of the nucleotide sequence at the desired time in thedesired tissue or part of the plant. Constitutive or inducible promotersare used. The transgene may also comprise other regulatory elements suchas for example translation enhancers or termination signals. In apreferred embodiment, the nucleotide sequence is the coding sequence ofa gene and is transcribed and translated into a protein. In anotherpreferred embodiment, the nucleotide sequence encodes an antisense RNA,a sense RNA that is not translated or only partially translated, at-RNA, a r-RNA or a sn-RNA.

Where more than one trait are introgressed into inbred maize lineNP2315, it is preferred that the specific genes are all located at thesame genomic locus in the donor, non-recurrent parent, preferably, inthe case of transgenes, as part of a single DNA construct integratedinto the donor's genome. Alternatively, if the genes are located atdifferent genomic loci in the donor, non-recurrent parent, backcrossingallows to recover all of the morphological and physiologicalcharacteristics of inbred maize line NP2315 in addition to the multiplegenes in the resulting maize inbred line. The genes responsible for aspecific, single gene trait are generally inherited through the nucleus.Known exceptions are, e.g. the genes for male sterility, some of whichare inherited cytoplasmically, but still act as single gene traits. In apreferred embodiment, a heterologous transgene to be transferred tomaize inbred line NP2315 is integrated into the nuclear genome of thedonor, non-recurrent parent. In another preferred embodiment, aheterologous transgene to be transferred to into maize inbred lineNP2315 is integrated into the plastid genome of the donor, non-recurrentparent. In a preferred embodiment, a plastid transgene comprises onegene transcribed from a single promoter or two or more genes transcribedfrom a single promoter.

In a preferred embodiment, a transgene whose expression results orcontributes to a desired trait to be transferred to maize inbred lineNP2315 comprises a virus resistance trait such as, for example, a MDMVstrain B coat protein gene whose expression confers resistance to mixedinfections of maize dwarf mosaic virus and maize chlorotic mottle virusin transgenic maize plants (Murry et al. Biotechnology (1993)11:1559–64). In another preferred embodiment, a transgene comprises agene encoding an insecticidal protein, such as, for example, a crystalprotein of Bacillus thuringiensis or a vegetative insecticidal proteinfrom Bacillus cereus, such as VIP3 (see for example Estruch et al. NatBiotechnol (1997) 15:137–41). In a preferred embodiment, an insecticidalgene introduced into maize inbred line NP2315 is a Cry1Ab gene or aportion thereof, for example introgressed into maize inbred line NP2315from a maize line comprising a Bt-11 event as described in U.S. Pat. No.6,114,608, which is incorporated herein by reference, or from a maizeline comprising a 176 event as described in Koziel et al. (1993)Biotechnology 11: 194–200. In yet another preferred embodiment, atransgene introgressed into maize inbred line NP2315 comprises aherbicide tolerance gene. For example, expression of an alteredacetohydroxyacid synthase (AHAS) enzyme confers upon plants tolerance tovarious imidazolinone or sulfonamide herbicides (U.S. Pat. No.4,761,373). In another preferred embodiment, a non-transgenic traitconferring tolerance to imidazolinones is introgressed into maize inbredline NP2315 (e.g a “IT” or “IR” trait). U.S. Pat. No. 4,975,374,incorporated herein by reference, relates to plant cells and plantscontaining a gene encoding a mutant glutamine synthetase (GS) resistantto inhibition by herbicides that are known to inhibit GS, e.g.phosphinothricin and methionine sulfoximine. Also, expression of aStreptomyces bar gene encoding a phosphinothricin acetyl transferase inmaize plants results in tolerance to the herbicide phosphinothricin orglufosinate (U.S. Pat. No. 5,489,520). U.S. Pat. No. 5,013,659, which isincorporated herein by reference, is directed to plants that express amutant acetolactate synthase (ALS) that renders the plants resistant toinhibition by sulfonylurea herbicides. U.S. Pat. No. 5,162,602 disclosesplants tolerant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides. The tolerance is conferred byan altered acetyl coenzyme A carboxylase (ACCase). U.S. Pat. No.5,554,798 discloses transgenic glyphosate tolerant maize plants, whichtolerance is conferred by an altered 5-enolpyruvyl-3-phosphoshikimate(EPSP) synthase gene. U.S. Pat. No. 5,804,425 discloses transgenicglyphosate tolerant maize plants, which tolerance is conferred by anEPSP synthase gene derived from Agrobacterium tumefaciens CP-4 strain.Also, tolerance to a protoporphyrinogen oxidase inhibitor is achieved byexpression of a tolerant protoporphyrinogen oxidase enzyme in plants(U.S. Pat. No. 5,767,373). Another trait transferred to inbred maizeline NP2315 confers tolerance to an inhibitor of the enzymehydroxyphenylpyruvate dioxygenase (HPPD) and transgenes conferring suchtrait are, for example, described in WO 9638567, WO 9802562, WO 9923886,WO 9925842, WO 9749816, WO 9804685 and WO 9904021. All issued patentsreferred to herein are, in their entirety, expressly incorporated hereinby reference.

In a preferred embodiment, a transgene transferred to maize inbred lineNP2315 comprises a gene conferring tolerance to a herbicide and at leastanother nucleotide sequence encoding another trait, such as for example,an insecticidal protein. Such combination of single gene traits is forexample a Cry1Ab gene and a bar gene.

Specific transgenic events introgressed into maize inbred line NP2315can be obtained through the list of Petitions of Nonregulated Statusgranted by APHIS as of 10-12-2000. For example, introgressed fromglyphosate tolerant event GA21 (9709901p), glyphosatetolerant/Lepidopteran insect resistant event MON 802 (9631701p),Lepidopteran insect resistant event DBT418 (9629101p), male sterileevent MS3 (9522801p), Lepidopteran insect resistant event Bt11(9519501p), phosphinothricin tolerant event B16 (9514501p), Lepidopteraninsect resistant event MON 80100 (9509301p), phosphinothricin tolerantevents T14, T25 (9435701p), Lepidopteran insect resistant event 176(9431901p).

The introgression of a Bt11 event into a maize line, such as maizeinbred line NP2315, by backcrossing is exemplified in U.S. Pat. No.6,114,608, and the present invention is directed to methods ofintrogressing a Bt11 event into maize inbred line NP2315 using forexample the markers described in U.S. Pat. No. 6,114,608 and toresulting maize lines.

Direct selection may be applied where the trait acts as a dominanttrait. An example of a dominant trait is herbicide tolerance. For thisselection process, the progeny of the initial cross are sprayed with theherbicide prior to the backcrossing. The spraying eliminates any plantwhich does not have the desired herbicide tolerance characteristic, andonly those plants that have the herbicide tolerance gene are used in thesubsequent backcross. This process is then repeated for the additionalbackcross generations.

This invention also is directed to methods for producing a maize plantby crossing a first parent maize plant with a second parent maize plantwherein either the first or second parent maize plant is a maize plantof inbred line NP2315 or a maize plant of inbred line NP2315 furthercomprising one or more single gene traits. Further, both first andsecond parent maize plants can come from the inbred maize line NP2315 oran inbred maize plant of NP2315 further comprising one or more singlegene traits. Thus, any such methods using the inbred maize line NP2315or an inbred maize plant of NP2315 further comprising one or more singlegene traits are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing inbred maize line NP2315 or inbred maize plants of NP2315 furthercomprising one or more single gene traits as a parent are within thescope of this invention. Advantageously, inbred maize line NP2315 orinbred maize plants of NP2315 further comprising one or more single genetraits are used in crosses with other, different, maize inbreds toproduce first generation (F1) maize hybrid seeds and plants withsuperior characteristics.

In a preferred embodiment, seeds of inbred maize line NP2315 or seeds ofinbred maize plants of NP2315 further comprising one or more single genetraits are provided as an essentially homogeneous population of inbredcorn seeds. Essentially homogeneous populations of inbred seed are thosethat consist essentially of the particular inbred seed, and aregenerally purified free from substantial numbers of other seed, so thatthe inbred seed forms between about 90% and about 100% of the totalseed, and preferably, between about 95% and about 100% of the totalseed. Most preferably, an essentially homogeneous population of inbredcorn seed will contain between about 98.5%, 99%, 99.5% and about 100% ofinbred seed, as measured by seed grow outs. The population of inbredcorn seeds of the invention is further particularly defined as beingessentially free from hybrid seed. The inbred seed population may beseparately grown to provide an essentially homogeneous population ofplants of inbred maize line NP2315 or inbred maize plants of NP2315further comprising one or more single gene traits.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which maize plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,kernels, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk, seeds and the like.

Duncan, Williams, Zehr, and Widholm, Planta (1985) 165:322–332 reflectsthat 97% of the plants cultured that produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262–265 reports several media additions that enhance regenerability ofcallus of two inbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao, et al., Maize Genetics CooperationNewsletter, 60:64–65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345–347(1987) indicates somatic embryogenesis from the tissue cultures of maizeleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture procedures of maize are described in Green and Rhodes,“Plant Regeneration in Tissue Culture of Maize,” Maize for BiologicalResearch (Plant Molecular Biology Association, Charlottesville, Va.1982, at 367–372) and in Duncan, et al., “The Production of CallusCapable of Plant Regeneration from Immature Embryos of Numerous Zea maysGenotypes,” 165 Planta 322–332 (1985). Thus, another aspect of thisinvention is to provide cells that upon growth and differentiationproduce maize plants having the physiological and morphologicalcharacteristics of inbred maize line NP2315. In a preferred embodiment,cells of inbred maize line NP2315 are transformed genetically, forexample with one or more genes described above, for example by using atransformation method described in U.S. Pat. No. 6,114,608, andtransgenic plants of inbred maize line NP2315 are obtained and used forthe production of hybrid maize plants.

Maize is used as human food, livestock feed, and as raw material inindustry. Sweet corn kernels having a relative moisture of approximately72% are consumed by humans and may be processed by canning or freezing.The food uses of maize, in addition to human consumption of maizekernels, include both products of dry- and wet-milling industries. Theprincipal products of maize dry milling are grits, meal and flour. Themaize wet-milling industry can provide maize starch, maize syrups, anddextrose for food use. Maize oil is recovered from maize germ, which isa by-product of both dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry. Industrial uses of maize include productionof ethanol, maize starch in the wet-milling industry and maize flour inthe dry-milling industry. The industrial applications of maize starchand flour are based on functional properties, such as viscosity, filmformation, adhesive properties, and ability to suspend particles. Themaize starch and flour have application in the paper and textileindustries. Other industrial uses include applications in adhesives,building materials, foundry binders, laundry starches, explosives,oil-well muds, and other mining applications. Plant parts other than thegrain of maize are also used in industry: for example, stalks and husksare made into paper and wallboard and cobs are used for fuel and to makecharcoal.

The seed of inbred maize line NP2315 or of inbred maize line NP2315further comprising one or more single gene traits, the plant producedfrom the inbred seed, the hybrid maize plant produced from the crossingof the inbred, hybrid seed, and various parts of the hybrid maize plantcan be utilized for human food, livestock feed, and as a raw material inindustry.

The present invention therefore also discloses an agricultural productcomprising a plant of the present invention or derived from a plant ofthe present invention. The present invention also discloses anindustrial product comprising a plant of the present invention orderived from a plant of the present invention. The present inventionfurther discloses methods of producing an agricultural or industrialproduct comprising planting seeds of the present invention, growingplant from such seeds, harvesting the plants and processing them toobtain an agricultural or industrial product.

DEPOSIT

Applicants have made a deposit of at least 2500 seeds of Inbred MaizeLine NP2315 with the American Type Culture Collection (ATCC), Manassas,Va., 20110-2209 U.S.A., ATCC Deposit No: PTA-4929. This deposit of theInbred Maize Line NP2315 will be maintained in the ATCC depository,which is a public depository, for a period of 30 years, or 5 years afterthe most recent request, or for the effective life of the patent,whichever is longer, and will be replaced if it becomes nonviable duringthat period. Additionally, Applicants have satisfied all therequirements of 37 C.F.R. §§1.801–1.809, including providing anindication of the viability of the sample. Applicants impose norestrictions on the availability of the deposited material from theATCC; however, Applicants have no authority to waive any restrictionsimposed by law on the transfer of biological material or itstransportation in commerce. Applicants do not waive any infringement ofits rights granted under this patent or under the Plant VarietyProtection Act (7 USC 2321 et seq.).

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somaclonal variants, variantindividuals selected from large populations of the plants of the instantinbred and the like may be practiced within the scope of the invention,as limited only by the scope of the appended claims.

1. Seed of maize inbred line designated NP2315, representative seed ofsaid line having been deposited under ATCC Accession No: PTA-4929.
 2. Amaize plant, or a part thereof, produced by growing the seed of claim 1.3. Pollen of the plant of claim
 2. 4. An ovule of the plant of claim 2.5. A maize plant, or a part thereof, having all the physiological andmorphological characteristics of the plant according to claim
 2. 6. Themaize plant, or a part thereof, produced from the maize plant accordingto claim 2 or 5, by transformation with a transgene that confers uponsaid maize plant or a part thereof tolerance to an herbicide.
 7. A maizeplant according to claim 6, wherein said herbicide is glyphosate,gluphosinate, a sulfonylurea herbicide an imidazolinone herbicide, ahydroxyphenylpyruvate dioxygenase inhibitor or a protoporphyrinogenoxidase inhibitor.
 8. A maize plant or a part thereof produced bytransforming the maize plant according to claim 2 or 5 with a transgenethat confers upon said maize plant or a part thereof, male sterility,insect resistance, disease resistance, bacterial resistance, fungalresistance or viral resistance.
 9. The maize plant according to claim 8,wherein said transgene is Bacillus thuringiensis Cry1Ab gene.
 10. Themaize plant according to claim 8, wherein said transgene furthercomprises a bar gene.
 11. A tissue culture of regenerable cells producedfrom themaize plant according to claim
 2. 12. The tissue cultureaccording to claim 11, wherein the regenerable cells are from a tissueselected from the group consisting of embryo, meristem, pollen, leave,anther, root, root tip, silk, flower, kernel, ear, cob, husk and stalk.13. A maize plant regenerated from the tissue culture of claim 11 or 12,wherein the regenerated plant has all of the morphological andphysiological characteristics of maize plant of inbred line NP2315, seedof said line having been deposited under ATCC Accession No: PTA-4929.14. A method for producing maize seed comprising crossing a first parentmaize plant with a second parent maize plant and harvesting theresultant maize seed, wherein said first or second parent maize plant isthe inbred maize plant of claim
 2. 15. The method according to claim 14,wherein the inbred maize plant is the female parent.
 16. The methodaccording to claim 14, wherein the inbred maize plant is the maleparent.